1. Field of the Invention
The present invention relates to functional, modified glucose-galactose binding proteins (GGBPs), that have a greater melting temperature (Tm) than a reference GGBP. The present invention also relates to nucleic acids encoding these thermostable GGBPs, as well as methods of using these thermostable GGBPs
2. Background of the Invention
A rapidly advancing area of biosensor development is the use of periplasmic binding proteins (PBPs), to accurately determine analyte, e.g., glucose, concentrations in biological samples. In particular, glucose-galactose binding proteins (GGBPs) are being employed as biosensors to measure analyte quantities in industrial and pharmacological physiological settings. PBPs are considered to be “reagentless” and can be used in a variety of settings including measuring glucose in monitoring diabetes, measuring amino acids in other metabolic diseases, such as histidase deficiency, as well as measuring arabinose during ethanol production from corn. Wild-type GGBPs, however, may not be the most ideal candidates for measuring or determining analyte concentrations for a variety of reasons. Biosensors comprising GGBPs would preferably be physically stable under conditions of use to generate a quantifiable signal on glucose binding. When the intended use is monitor in vivo glucose concentrations in diabetics the proteins would preferably be stable at physiological temperatures. Additionally, the GGBPs would preferably have enhanced stability for sensor manufacturing, shipping and storage, which could potentially streamline and enable the protein and sensor materials to be fabricated at ambient temperature. This manufacturing process could include high-temperature sterilization procedures for use in a clinical setting. Exposure to high temperatures, however, may denature the protein, rendering the GGBPs useless for their intended purpose. Thus there is a need for GGBPs that are able to withstand higher temperatures while remaining active such that they can bind analyte and be used in biosensors in a variety of higher temperature settings.